The present invention is directed to automated analytical apparatus to detect the time of formation of fibrin clots in human or animal blood plasma by the prothrombin time (PT) test, the activated partial thromboplastin time (APTT) test, thrombin time test (TT) or other clotting factor tests and assays. The invention also has utility in other automated testing applications in addition to blood coagulation, including but not limited to clinical chemistry, serology and the like.
Methods of detecting the formation of fibrin clots date back to the late 1870's. Such methods were manual. For example, in one test a white horse hair was drawn through a blood specimen. The endpoint of the clotting time was the point where shreds of fibrin could be visually detected on the hair. By 1910, an electrical apparatus called a "coaguloviscosimeter" was developed which directly measured the change in viscosity of a blood sample as it clotted. The apparatus provided a direct indication of voltage which could be plotted against clotting time.
In the early 1920's, rudimentary photoelectric techniques were developed to detect variations in light transmissivity of a blood sample during clotting. An apparatus termed a "nephelometer" was devised which consisted of a light source that furnished constant illumination to the sample. During coagulation of the sample, variations in the optical transmissivity of the sample were registered by a thermopile connected to a sensitive galvanometer. By reading the movements of the galvanometer needle, transmissivity values could be plotted against elapsed time.
In the mid 1930's, investigations of the coagulation of blood plasma using more sophisticated photoelectric techniques were conducted. It was noted that an increase in density occurs as blood coagulates. The possibility of detecting this change by photoelectric techniques was investigated. This led to the development of an instrument which displayed increasing density of the sample as a gradual change in the voltage displayed by a galvanometer. In addition, a water bath was used to maintain the blood sample at 37.degree. C.
Early photoelectric systems were limited to one specimen at a time, and there was no way to compensate for differences in plasma density and color variations from specimen to specimen. There was also no common reference point.
Today, five automated clotting time measurement techniques are in use: (1) electromechanical; (2) clot elasticity; (3) fibrin adhesion; (4) impedance; and (5) optical density. The principal electromechanical method in use today involves the use of a "fibrin switch" in which the physical formation of fibrin strands in a reaction mixture serves to complete an electrical circuit between two electrodes, thus stopping a timer. There are a number of limitations to "fibrin switch" systems. Clot formation cannot continually be observed. They are prone to cross-contamination and mechanical failure. An operator must clean the electrodes, exposing the operator to the risk of infection.
Clot elasticity analyzers consist of a pin, with an attached mirror, which rotates in a stainless steel cuvette which contains the plasma sample. Light is directed onto the mirror and is reflected from the mirror onto photosensitive film. As the clot develops, the elasticity of the sample changes, changing the mirror position and altering the pattern of light projected on the film. Either plasma or whole blood may be used, but whole blood testing is inferior to plasma testing because (1) extraneous factors in whole blood may affect test results, (2) test time is much longer, and (3) the test is much less specific.
In fibrin adhesion systems, a filament moves through the sample, and the clot adheres to the filament as the clot forms. The clot, which becomes attached to and moves with the filament, interrupts a light beam to initiate end point detection. Either plasma or whole blood may be used with this system.
In impedance detection systems, a special sensing probe is moved through a sample. As the clot forms, the probe movement is impeded. More energy is required to maintain the same degree of movement of the probe through the sample. The instrument displays a recording of the amount of energy required to keep the probe in motion. The amount of energy can be related to clotting time.
Optical density detection systems operate on the principle that an increase in density of the coagulating plasma will decrease the transmission of light through the sample. The test sample is placed in a transparent sample cuvette and reacted with a test reagent. Light is directed through the reacted sample. A typical test reagent used in coagulation testing is a biological substance called thromboplastin, derived from brain tissue of rabbits. Such reagents are delicate and expensive. Advantages of optical systems include (1) no contact with sample, no cross-contamination, and no contact activation by agitating the specimen; (2) continuous observation of clot formation, which yields increased reproducibility of test results; (3) a consistent end point; and (4) ease of automation, thereby minimizing human error.
Modern optical systems no longer depend on an absolute optical density change or a direct voltage reading from a photocell. Instead modern systems operate on the first or second derivative of the photocell voltage. Thus, modern systems are independent of initial optical density or color of the sample.
Most optical detection systems in use today utilize lines of sight transverse to the sample cuvette. A few use lines of sight axial to the sample cuvette. In transverse line-of-sight detectors, a light source and photodetector are placed on diametrically opposite sides of the sample cuvette. Such transverse line-of-sight systems typically require large volumes of plasma and reagent, because it is necessary, for accurate detection, to direct the light through approximately the central third of the sample. Since relatively large amounts of expensive test reagent are required, transverse line-of-sight detectors are expensive to operate.
In axial line-of-sight systems, a light source is located above the sample cuvette and the photodetector is located underneath the cuvette. With axial line-of-sight systems, the optics must compensate for the meniscus at the surface of the sample. Also, the volume of sample and reagent must be controlled to extremely close tolerances, since differences in liquid depth in the cuvette could alter the test results. Moreover, when the sample and the reagents are added to the cuvette, frothing of the sample may occur, resulting in a number of bubbles on and below the surface of the sample. The bubbles will lead to false readings and unreliable results.
No existing equipment integrates plasma separation from whole blood as part of the equipment in order to perform testing on plasma only.
It is an object of the present invention to provide an automated analytical apparatus to detect the time of formation of fibrin clots in human or animal blood plasma.
It is also an object of the invention to integrate plasma separation from whole blood as part of the automated analytical apparatus in order to perform testing on plasma only. By integrating plasma separation from whole blood as part of the apparatus, a separate prior and necessary centrifugation operation is eliminated. Accordingly, throughput of test samples can be increased. In addition, freshly-filtered plasma yields more accurate test results than plasma obtained by centrifugation.
It is also an object of the invention to provide an analytical apparatus which is "user friendly" and eliminates manual operations.
It is also an object of the invention to provide a disposable sample cell for receiving samples of fluid to be tested. Making the sample cell disposable reduces the possible risk of infection from blood samples.